Influenza A and B viruses have tremendous socioeconomic consequences, for influenza continues to occur in regular epidemics and occasional pandemics and is a leading cause of morbidity and mortality. The 2009 H1N1 pandemic was a wake-up call that influenza is still a continuing threat. Of continuing great concern today is the highly virulent avian H5N1 influenza virus that is still circulating among birds and transmitting to humans in parts of Far East Asia. Each year in the USA, in the absence of the introduction of pandemic influenza, seasonal influenza kills 36,000 people, hospitalizes 114,000 and causes 70 million missed work days and 38 million lost school days. It is estimated the loss to the economy is $3-15 billion. In the years of introduction of pandemic influenza virus, 1957 and 1967, approximately 70% of the US population was infected and in 1918/19 the estimate of death associated with Spanish influenza range from 20-40 million (1 in 100 people). Thus, there remains a need for efficient control of influenza virus by a new generation of vaccines or specific chemotherapeutics agents. The aim of this grant application is to study the mechanism of influenza virus assembly. The direct relevance of this research to public health is that knowledge gained on the assembly mechanism of influenza virus provides new targets for anti-viral drug development. Among the least understood aspects of influenza virus replication are the events that allow the formation of virus particles and the pinching off o these particles by membrane fission. Recently, we have found a new role for the influenza virus M2 proton-selective ion channel protein in mediating virus budding. Our data indicate that an amphipathic helix (AH) in the M2 cytoplasmic tail mediates the final steps of budding for influenza viruses causing membrane scission and bypassing the need for using host cell proteins, as needed for budding of some other enveloped viruses e.g. HIV. We will perform a rigorous analysis of the function of the M2 cytoplasmic tail AH by generating a defined range of mutants in the AH and creating novel influenza viruses by reverse genetics. We will analyze the mutant viruses for changes in properties of the M2 protein function. We will analyze and reconstitute influenza virus budding in vitro and in vivo using assays that are well established in the field of membrane biophysics but are under utilized in the field of virology and that we have developed in our laboratory. We will analyze virus assembly in vivo by isolating membrane sheets. This permits, by using immuno-gold EM, examination of the temporal and spatial relationships between integral and membrane-associated viral proteins. This will provide major insight into the organization of the region of the plasma membrane involved in virus budding. PUBLIC HEALTH RELEVANCE: Influenza virus remains an important viral pathogen of significant medical importance with pandemics occurring in 1918, 1957 and 2009: the 2009 H1N1 pandemic was a wake-up call that influenza is still a continuing threat and justifying that influenza virus is classified as a Select Agent Category C, because of its ability to cause great morbidity and mortality. Thus, there remains a need for efficient control of influenza virus by a new generation of vaccines or specific chemotherapeutics agents. The aim of this grant application is to study the mechanism of influenza virus assembly and the direct relevance of this research to public health is that knowledge gained on the assembly mechanism of influenza virus provides new targets for anti-viral drug development.